Ex vivo microscopy is an emerging set of imaging modalities intended to enable histological imaging of whole tissue specimens at the point-of-procedure. Ex vivo microscopy holds much promise for enabling rapid verification of cancer biospecimen quantity and quality at the time of specimen acquisition. Two areas in which such technologies would be extremely helpful to pathologists are in 1) screening of biopsies collected during a biopsy procedure with the aim of providing guidance for collection of additional tissues, and 2) non-destructively assessing and verifying cancer content in biospecimens collected for banking and downstream molecular analysis. In prostate cancer diagnosis, for example, 70-80% of the 1 million biopsies collected for diagnosis each year are ultimately found to be benign on permanent histopathology review. For tissue banking, the major limiting pre- analytical factor for downstream molecular and genetic analysis is the amount of cancerous tissue present in the banked biopsies. However, currently available in-procedure pathology methods to assess biopsy cancer content (such as frozen section analysis) are too destructive, slow, expensive, and labor-intensive to be widely adopted. Thus, there is a missing opportunity to improve the efficiency and efficacy of diagnostic biopsy, and to guide sample collection and improve cancer biospecimen quality in tissue banking, by non-destructive histological verification. Until now, methods for ex vivo microscopy with the resolution and contrast needed for accurate biopsy verification have been too slow and expensive to realistically be adopted for clinical workflows involving multiple biopsies (16-20 in the case of prostate diagnostic biopsy). Further, the contrast methods (typically topical fluorescent staining with a single dye) have left much to be desired in terms of providing images that leverage the extensive training of pathologists. We have recently developed a set of technologies to address these limitations in an effort to ease adoption. The first is a rapid microscopic optical sectioning scanner, based on video-rate structured illumination microscopy that provides high-resolution, high-contrast images of fluorescently-stained biopsies in seconds at the point-of-procedure. This technology has been optimized for ease-of-use in the pathology workflow, including novel autofocus functions that enable fully automated imaging with minimal user intervention. The second is our recent development of a dual-color fluorescent staining procedure that exactly recapitulates the H&E staining used in standard histopathology. In this R33 Advanced Development project, we will build on these developments to validate VR-SIM as a practical, adoptable ex vivo microscopy solution for point-of-procedure biospecimen verification. In this project we will develop a dual-color version of the VR-SIM system optimized for our fluorescent H&E analog stain, optimize the system for balancing image resolution, contrast, and image acquisition speed, and develop a robust secure web-based multi-resolution image viewer for fast and facile telepathology of the VR-SIM images from any computer with a web browser. The project will culminate in a direct validation challenge of VR-SIM versus frozen section analysis for point-of-procedure biopsy verification in 200 fresh prostate core biopsies.